Electron microscopes are renowned for their ability to peer down into the hidden world of the very small. Trouble is, these tools only produce images in black and white. A new technique that took 15 years to develop finally overcomes this optical limitation, producing the first ever multicolor electron microscope images.

“It’s a bit like when you first see a color photograph after having only known black and white.”

A research team from the University of California, San Diego is the first to create a multicolor electron microscope, allowing for three colors at a time (green, red, and yellow). Technically speaking, the microscope is not producing “true” color images, but rather a false-color visualization of key features found within microscopic objects, such as cells. Importantly, the colors are not “added” after the fact—they’re genuinely indicative of discrete biological components.

The project was headed by Mark Ellisman and Roger Tsien (a 2008 Chemistry Nobel Prize laureate who died unexpectedly this past summer). The team used the new technique to capture color photographs of cellular membranes and the synaptic connections between brain cells.

“It’s a bit like when you first see a color photograph after having only known black and white,” noted first author Stephen Adams, a UCSD chemist, in a statement. “[For] the last 50 years or so, we’ve been so used to monochrome electron micrographs that it’s now hard to imagine that we could go back.”

Conventional electron microscopes form images by transmitting electron beams through an object, like a biological specimen. This allows for the creation of a detailed monochrome image, but because the microscope is shooting out electrons, and not colored light, there’s a definite absence of color.

A Golgi (an organelle found in most eukaryotic cells) is shown in green, and a cell membrane appears in red. (Image: Adams et al./Cell Chemical Biology 2016)

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To create the colorized scans, the researchers fitted a special detector on a conventional electron microscope. The researchers then selectively “painted” structures such as proteins, membranes, and cells with various “rare earth” metals, including lanthanum, cerium and praseodymium in the form of a chemical solution. When these specimens were scanned by the modified microscope, the stream of electrons lost by the metallic elements were interpreted as color.

“A transmission electron microscope can distinguish each of these metals by electron energy-loss to give elemental maps of each that can be overlaid in color on the familiar monochrome electron micrograph,” explained Adams. “Each color highlights a different component of the cellular ultrastructure.”

The process is actually quite similar to fluorescence microscopy, in which glowing proteins are added to a specimen. But with electron microscopy, the images are observed at far higher resolutions.

Using the new technique, the researchers used the multicolor electron microscope to visualize a pair of brain cells sharing a single synapse. The team also demonstrated how peptides (short chains of amino acids) are able to penetrate through a cell membrane.

The researchers say the new microscope will help biologists to distinguish cellular compartments, track proteins, and tag individual cells. Looking ahead, the team is hoping to improve the process and produce images with three or more colors.